A placenta allows eutherian mammals to give birth to progeny that are already developmentally advanced. A wide range of placental phenotypes exist across taxa to ensure adequate nutrient transfer from mother to offspring. The human placenta forms particularly intimate associations with maternal tissues through two types of cells: 1) syncytiotrophoblast (STB) cells, the fetally-derived trophoblast (TB) cell subset that coats the surface of the villous placenta, and 2) extravillous TB (EVCTB), which invade deeply, interact with maternal decidual immune cells, and remodel the spiral arteries. Both TB subsets are in direct contact with maternal blood and maternal immune cells and both are derived from an ill-defined, stem-cell like, cytotrophoblast (cytoTB) cell subpopulation. Although many adverse pregnancy outcomes may result from poor placental development, the study of the earliest stages of placental development cannot be performed in humans for ethical reasons. For example, alterations in STB development and turnover have been implicated in placental disease, including preeclampsia (PE), the leading single cause of premature birth. STB is the source of placental debris, cytokines, and pro-inflammatory and anti-angiogenic proteins, which become elevated in mothers diagnosed with PE. Appropriate in vitro models of early placental development are essential if we are to better understand and treat diseases of poor placentation. The first goal of this proposal is to generate STB and its mononucleated precursors from human embryonic stem cells (hESC) that have been treated with BMP4 (BMP-hESC) and inhibitors of activin and FGF2 signaling. We will use this system as a model to study STB emergence, especially the process of cell fusion and cell death, as well as intrinsic and extrinsic factors that influence these transitions. This project has high significance because STB forms the major interface with maternal blood perfusing the placenta and is responsible for the production of endocrine factors such as hCG and exchange of gases, nutrients and waste products The second goal is to demonstrate the utility of the BMP-hESC model to examine the molecular events controlling STB formation and lifespan, with an emphasis on the roles of transcription factors, such as GCM1 & GATA2, fusogenic proteins such syncytin-1 & -2 (ERVW1, ERVFRD-1, respectively) and a presently little understood HERV envelope gene product (ERV-Fb1) that inhibits cell-cell fusion. A third goal is to assess whether some features of PE can be unearthed in the BMP-hESC model. Our hypothesis is that TB from a sub-group of conceptuses whose mothers develop PE early in their pregnancies is unusually sensitive to high oxygen tensions. Since STB will only begin to encounter well-oxygenated maternal blood after the uteroplacental arteries open towards the end of the first trimester of pregnancy, the oxygen hypersensitivity of cells derived from PE placentas may cause them to turnover at an accelerated rate and shed cell contents and debris into the circulating maternal blood more quickly than normal STB. We will test this hypothesis in vitro using iPSCs derived from the umbilical cords of babies born to mothers with severe preeclampsia and gestational age-matched control iPSCs.